Negatively charged membrane

ABSTRACT

The present invention provides, in certain embodiments, a negatively charged microporous membrane comprising a porous substrate and a crosslinked coating having fixed negative charges. The crosslinked coating can be prepared, e.g., from a polymerized composition comprising an unsaturated monomer having an anionic group, an N-(hydroxymethyl)- and/or N-(alkoxymethyl)-acrylamide, a hydrophilic unsaturated monomer, and an initiator. The present invention further provides, in some embodiments, a negatively charged microporous membrane comprising a porous substrate and a crosslinked coating prepared from a polymerized composition comprising an unsaturated monomer having an anionic group, an N-(hydroxymethyl)- or N-(alkoxymethyl)-acrylamide, a polysaccharide, and an initiator. The membranes of the present invention are suitable for use in ion exchange chromatography, for example, in the separation and purification of positively charged species such as proteins.

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/914,165, filed on Aug. 24, 2001 which is a 371 ofPCT/US00/04745, filed on Feb. 25, 2000. This application claims priorityfrom U.S. Provisional Patent Application No. 60/128,668, filed on Feb.25, 1999, the disclosure of which is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

[0002] The present invention generally relates to negatively chargedmembranes, and in particular to negatively charged membranes comprisinga porous substrate and a crosslinked coating. The membranes find use inthe treatment of fluids containing positively charged species such asproteins, e.g., in ion-exchange chromatography.

BACKGROUND OF THE INVENTION

[0003] Negatively charged ion-exchange membranes have been proposed forthe separation and/or purification of biomolecules such as proteins,amino acids, and nucleic acids. For the ion exchange membrane to performeffectively in the above applications, the membrane should satisfyseveral important parameters. For example, the membrane should exhibithigh rates of fluid flow. The membrane should have high dynamic bindingcapacity for biomolecules, and should be capable of selectively bindingthe biomolecules, which have different surface charges. The membraneshould, therefore, have low non-specific binding, e.g., resulting fromhydrophobic interactions. The membrane should withstand high treatmentfluid velocities. The preparation of the membrane should not involvechemistries and processes that are cumbersome to practice. Some of thecation exchange membranes known heretofore suffer from the failure tosatisfy one or more of the parameters set forth above.

[0004] Accordingly, there exists a need for a cation exchange membranethat exhibits high rates of fluid flow. There further exists a need fora cation exchange membrane that has high dynamic binding capacity andselectivity for biomolecules. There further exists a need for a membranethat has low non-specific binding or low binding that results fromhydrophobic interactions. There further exists a need for a membranethat can withstand high fluid flow velocities. There further exists aneed for a membrane that involves preparation chemistries and/orprocesses that are not cumbersome to practice.

[0005] These advantages of the present invention, as well as additionalinventive features, will be apparent from the description of theinvention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1 depicts the breakthrough curve for lysozyme obtained on anembodiment membrane of the present invention. The x-axis represents thefiltration time, and the y-axis represents the absorbance of thefiltrate at 280 nm and is indicative of the concentration of theprotein. See Example 2 for additional details.

[0007]FIG. 2 depicts the breakthrough curve for lysozyme obtained onanother embodiment membrane of the present invention. The x-axis andy-axis are as described in FIG. 1. See Example 3 for additional details.

[0008]FIG. 3 depicts the breakthrough curve for lysozyme obtained onanother embodiment membrane of the present invention. The x-axis andy-axis are as described in FIG. 1. See Example 4 for additional details.

BRIEF SUMMARY OF THE INVENTION

[0009] Many of the foregoing needs have been fulfilled by the presentinvention which provides a negatively charged microporous membranecomprising a porous substrate and a crosslinked coating havingnegatively charged groups. In a preferred embodiment, the membrane canbe prepared from a polymerized composition comprising an unsaturatedmonomer having an anionic group, at least one or more N-(hydroxyalkyl)-and/or N-(alkoxyalkyl)-acrylamides, and a hydrophilic unsaturatedmonomer.

[0010] The present invention further provides a negatively chargedmicroporous membrane comprising a porous substrate and a crosslinkedcoating prepared from a hydroxyl-rich material such as a polysaccharideand a polymerized composition comprising an unsaturated monomer havingan anionic group, at least one or more N-(hydroxymethyl)- and/orN-(alkoxymethyl)-acrylamides, and an initiator.

[0011] The present invention further provides a negatively chargedmicroporous membrane having a protein binding capacity of about 25 mg/mllysozyme or more comprising a porous substrate and a crosslinked coatingthat provides a fixed negative charge. The present invention furtherprovides a negatively charged microporous membrane comprising a poroussubstrate and a crosslinked coating comprising a polymer having anionicgroups and amide-amide and amide-ester crosslinks.

[0012] The membranes of the present invention are advantageously free ofcovalent bonds or grafts with the substrate.

[0013] The present invention further provides a process for preparing anembodiment of the membrane comprising coating a porous substrate with apolymerized composition comprising an anionic group and curing themembrane. The membrane can be optionally washed or leached to removeextractable residue therein.

[0014] The present invention further provides devices, e.g., filterdevices, chromatographic devices, macromolecular transfer devices, andmembrane modules comprising the membranes of the present invention. Thepresent invention further provides a process for separating a positivelycharged material such as positively charged atoms, molecules, andparticulates, and, preferably, biomolecules, from a fluid, the processcomprising placing the fluid in contact with the negatively chargedmicroporous membrane so as to adsorb the positively charged material tothe membrane. The present invention further provides a process fortreating a fluid containing positively charged materials comprisingcontacting the fluid with a membrane of the present invention andselectively releasing the positively charged materials. The presentinvention further provides a process for transferring macromoleculesfrom a device or element such as an electrophoresis gel comprisingcontacting the gel with the membrane of the present invention andtransferring the biomolecules to the membrane.

[0015] While the invention has been described and disclosed below inconnection with certain preferred embodiments and procedures, it is notintended to limit the invention to those specific embodiments. Rather itis intended to cover all such alternative embodiments and modificationsas fall within the spirit and scope of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention provides embodiments of negatively chargedmembranes having high charge density, high water flow rates, highdynamic protein binding capacity, and low non-specific protein bindingcapacity. The membranes of the present invention find use in cationexchange chromatography and in the separation and/or purification ofcharged species, especially biomolecules such as proteins.

[0017] The present invention provides, in some embodiments, a negativelycharged microporous membrane comprising a porous substrate and acrosslinked coating having anionic groups. The crosslinked coating canbe prepared from a polymerized composition comprising an unsaturatedmonomer having an anionic group, at least one or more N-(hydroxyalkyl)-or N-(alkoxyalkyl)-acrylamides, a hydrophilic unsaturated monomer. Thecomposition can further include an initiator. In preferred embodiments,the N-(hydroxyalkyl)- or N-(alkoxyalkyl)-acrylamide is one wherein thealkyl moiety has 4 or less carbon atoms, and more preferably the alkylmoiety is methyl.

[0018] In certain embodiments, the membrane comprises a porous substrateand a crosslinked coating prepared from a polymerized compositioncomprising an unsaturated monomer having an anionic group, at least oneor more N-(hydroxymethyl)- and/or N-(alkoxymethyl)-acrylamides, ahydroxyl-rich material such as a polysaccharide, and optionally aninitiator. The present invention further provides a negatively chargedmicroporous membrane comprising a porous substrate and a crosslinkedcoating comprising a polymer having anionic groups and amide-amide andamide-ester crosslinks.

[0019] The membrane of the present invention contains fixed anionicgroups. The anionic group can be any negatively charged group—sulfonic,carboxylic, phosphonic, and the like, preferably sulfonic or carboxylicacid groups. The coating composition comprises an unsaturated monomerhaving an anionic group. Any suitable unsaturated monomer—vinyl, vinylaromatic, acrylic, or other monomer can be used.

[0020] The unsaturated monomer preferably is an acrylic monomer. Theacrylic monomer can be an acrylate or an acrylamide. The acrylic monomeris preferably an acrylamide. The term “acrylamide” herein refers tounsubstituted as well as substituted monomers having a —C═C—(C═O)—N—moiety. The nitrogen and the C═C carbon atoms can be attached tohydrogen or other nonpolar substituents. An example of such substituentsis alkyl. Thus, the substituted acrylamide can be alkylacrylamide. Theterm “alkyl” herein refers to an alkyl group having from 1 to about 10carbon atoms, preferably from 1 to about 6 carbon atoms, and morepreferably from 1 to about 3 carbon atoms. An example of an acrylamidemonomer having a sulfonic acid group is acrylamido-N-alkylsulfonic acid,preferably 2-acrylamido-2-methyl-1-propanesulfonic acid. Preferredexamples of acrylic monomers having a carboxylic acid group are3-acrylamido-3-methylbutanoic acid (AMBA), 2-acrylamidoglycollic acid,and β-carboxyethyl acrylate.

[0021] In certain embodiments, the coating composition comprises ahydrophilic unsaturated monomer, e.g., a nonionic hydrophilicunsaturated monomer. Any suitable hydrophilic unsaturated monomer can beused, preferably an acrylic monomer. The monomer contains one or morepolar groups that contribute hydrophilicity. Examples of such groupsinclude hydroxy, alkoxy, hydroxyalkyl, and amido. Preferred hydrophilicgroups are hydroxyl and hydroxyalkyl. Thus, preferred hydrophilicacrylic monomers are hydroxyacrylic and hydroxyalkylacrylic. The acrylicmonomer can be an acrylate ester or an acrylamide. An example of apreferred hydroxyalkyl acrylate monomer is hydroxypropyl methacrylate.

[0022] The coating composition comprises a crosslinking agent. Anysuitable crosslinking agent known to those of ordinary skill in the artcan be used. Preferred crosslinking agents includeN-(alkoxymethyl)acrylamide and N-(hydroxymethyl)acrylamide.N-(isobutoxymethyl)acrylamide is further preferred.

[0023] The coating composition preferably comprises an initiator. Anysuitable initiator—free radical initiator, photoinitiator, and the like,can be used. A free radical initiator is preferred. An example of asuitable free radical initiator is a persulfate such as ammoniumpersulfate.

[0024] Without being bound to any particular theory, it is believed thatthe use of the three monomers in certain embodiments contributes toincreased spatial separation of charges. Thus, it is believed that thedistance between the anionic groups is increased. This increaseddistance disfavors association of the anionic groups. Accordingly,inter- and/or intra-chain association of anionic groups is reducedcompared to a system wherein only an anionic monomer and a crosslinkingmonomer are employed, particularly in a two monomer system wherein ahydrophilic or hydroxyl-rich material such as a polysaccharide is notemployed. The reduced association makes the negatively charged groupsavailable for interaction with positively charged molecules in thetreated fluid. This results, for example, in enhanced dynamic proteinbinding capacity.

[0025] The membrane according to some embodiments is made from a coatingcomposition that includes a hydroxyl-rich material, which may be a smallmolecule or a polymer having a plurality of hydroxyl groups, e.g., two,three, four or more hydroxyl groups per molecule. Examples ofhydroxyl-rich materials include polysaccharides and polyvinyl alcohol,preferably polysaccharides. Without being bound to any particularmechanism, it is believed that the hydroxyl groups of the hydroxyl-richmaterial involve in hydrogen bonding with the fluid. The saccharide ringrepeat units exert steric effects. Operation of one or both of thesemechanisms results in increased charge separation among the anionicgroups. The increased charge separation is believed to reduce anionassociation and facilitate interaction between the anionic sites and thepositively charged species in the treated fluid.

[0026] Any suitable polysaccharide can be used, preferably a watersoluble polysaccharide. An example of a preferred polysaccharide isdextran. The molecular weight of the dextran is below about 40,000,000,e.g., from about 10,000 to about 2,000,000, preferably from about 10,000to about 500,000, and more preferably from about 10,000 to about300,000. Particular examples of suitable molecular weights include110,000 and 148,000.

[0027] The coating composition of certain embodiments can be prepared bycombining and polymerizing the acrylic monomer having an anionic group,the nonionic hydrophilic monomer, a crosslinking agent, and theinitiator. In some embodiments, the coating solution is prepared bycombining and polymerizing the acrylic monomer having an anionic group,the polysaccharide, the crosslinking agent, and the initiator.

[0028] The polymerization can be carried out in a solvent, preferably inwater or water/methanol solution. The polymerization is preferablystopped prior to the formation of a gel or excessive crosslinking. Theviscosity of the polymerization solution can be monitored to control thedegree of polymerization. The polymerization is carried out for anysuitable length of time, e.g., for about 4 hours or more. According tocertain embodiments, the polymerization is carried out for a period offrom about 4 hours to about 5 hours. According to certain otherembodiments, the polymerization is carried out for a period of fromabout 16 hours to about 24 hours. The viscosity of the solution istypically below about 2000 cps, e.g., from about 50 cps to about 500cps, preferably from about 50 cps to about 500 cps, and more preferablyfrom about 100 cps to about 500 cps. According to certain embodiments,the viscosity is from about 100 cps to about 250 cps.

[0029] The polymerization solution can contain the anionic acrylicmonomer (A), the crosslinking agent (B), and the non-ionic hydrophilicmonomer (C) in a suitable ratio. The percentage of each monomer (A, B,or C) can be from about 0.1 to 30% by weight, preferably from about 0.1to 20% by weight.

[0030] It is believed that the crosslinked coating comprises amide-estercrosslinks that form as a result of the reaction of the nonionichydrophilic monomer with the crosslinking agent. For example, thesebonds form as a result of the reaction of the hydroxyl groups in thehydroxyalkyl acrylate with the N-(isobutoxymethyl)-acrylamide. Inaddition, amide-amide crosslinks also form as a result of the reactionbetween two N-(isobutoxymethyl)acrylamide monomers. For example, theamide-ester crosslink can have the formula:

—C(═O)O—R—NH—C(═O),

[0031] wherein R is divalent radical, preferably an alkoxyalkyl radical,and more preferably —CH₂—CH₂—CH₂—O—CH₂—. The amide-amide crosslink canhave the formula:

—C(═O)NH—R—NH—C(═O),

[0032] wherein R is divalent radical, preferably an alkoxyalkyl radical,and more preferably —CH₂—O—CH₂—.

[0033] The coating solution contains the anionic polymer prepared asabove and, optionally, a polysaccharide, preferably a dextran. Theanionic polymer and the polysaccharide can be present in the coatingsolution in the ratio of from about 100:1 to about 1:100, preferablyfrom about 10:1 to about 1:10, and more preferably from about 5:1 toabout 1:5.

[0034] The coating solution contains the anionic polymer and, optionallydextran, in an amount of from about 0.01% to about 15% by weight,preferably from about 0.1% to about 10% by weight, and more preferablyfrom about 0.5% to about 5% by weight of the coating solution. Forexample, the coating solution can contain 4.5% by weight of polymer and1.5% by weight of dextran.

[0035] The pH of the coating solution can be adjusted suitably. Forexample, the pH of the coating solution containing a carboxylatedpolymer can be adjusted to about 3.0 to about 4.0 and preferably about3.75. The pH of the coating can be adjusted by the addition of an acidor base. An example of a suitable base is 2N NaOH aqueous solution.

[0036] The coating solution is coated on a porous substrate, preferablya hydrophilic substrate. The hydrophilic porous substrate can be made ofany suitable material; preferably, the substrate comprises a polymer.Examples of suitable polymers include polyaromatics, polysulfones,polyolefins, polystyrenes, polycarbonates, polyamides, polyimides,fluoropolymers, cellulosic polymers such as cellulose acetates andcellulose nitrates, and PEEK. Aromatic polysulfones are preferred.Examples of aromatic polysulfones include polyethersulfone, bisphenol Apolysulfone, and polyphenylsulfone. Polyethersulfone is particularlypreferred. The porous substrate can have any suitable pore size, forexample, a pore size of below about 10 μm, e.g., from about 0.01 μm toabout 10 μm, preferably from about 0.1 μm to about 5 μm, and morepreferably from about 0.2 μm to about 5 μm. The porous substrate can beasymmetric or, in a preferred embodiment, symmetric.

[0037] The porous substrate can be prepared by methods known to those ofordinary skill in the art. For example, the porous substrate can beprepared by a phase inversion process. Thus, a casting solutioncontaining the polymer, a solvent, a pore former, a wetting agent, andoptionally a small quantity of a non-solvent is prepared by combiningand mixing the ingredients, preferably at an elevated temperature. Theresulting solution is filtered to remove any impurities. The castingsolution is cast or extruded in the form of a sheet or hollow fiber. Theresulting sheet or fiber is allowed to set or gel as a phase invertedmembrane. The set membrane is then leached to remove the solvent andother soluble ingredients.

[0038] The porous substrate can be coated with the coating solution bymethods known to those of ordinary skill in the art, for example, by dipcoating, spray coating, meniscus coating, and the like. Dip coating, forexample, can be carried out as follows. The substrate is immersed in thesolution for a given period of time sufficient to insure complete orsubstantially complete coating of the pore walls. The immersion time canbe from about 1 second to 1.0 minute, preferably from about 0.1 minutesto about 0.5 minutes, and more preferably from about ⅙ minute to about ⅓minute. Following the immersion, the excess coating solution on thesubstrate is removed by allowing it to drain under gravity or by the useof a squeegee or air knife. The resulting coated substrate is cured toeffect the curing or crosslinking of the coating composition.

[0039] Thus, the membrane can be cured below 150° C., e.g., at atemperature of from about 60° C. to about 130° C., and preferably at atemperature of from about 80° C. to about 130° C., for a suitable periodof time, which can range from about 5 minutes to about 120 minutes andpreferably from about 5 minutes to about 60 minutes. According tocertain embodiments, the membrane is cured at a temperature of fromabout 120° C. to about 125° C. for a period of from about 20 minutes toabout 30 minutes.

[0040] The resulting membrane can be washed to leach out any extractablein the membrane. Certain embodiments of the membrane, particularly amembrane having carboxyl functionality, are washed or leached in a basicsolution, preferably at a pH of from about 8 to about 12. The leachingliquid can be prepared by adding a base such as sodium hydroxide, sodiumcarbonate, or sodium bicarbonate. The base can be added as a solid or asolution. Particular examples of pH's of the leaching liquid are about11.9, about 11.4, and about 8.1. These pH's can be achieved by the useof, e.g., a 2N NaOH solution, sodium carbonate, or sodium bicarbonate.

[0041] Illustratively, a carboxylated membrane can be washed or leachedat a temperature of from about 37° C. to about 93° C. or higher andpreferably from about 54° C. to about 73° C. or higher. A sulfonic acidcontaining membrane can be washed or leached at a temperature of fromabout 54° C. to about 93° C. or higher. Embodiments of the membrane alsocan be leached in hot deionized water, e.g., deionized water held above73° F. The washing or leaching can be carried out for a suitable lengthof time, for example, for about 20 to about 30 minutes or more.According to certain embodiments of the membrane, the washing orleaching can be carried out for about 1 hour or more. The resultingmembrane is then dried in air or in an oven to remove the water.

[0042] The present invention provides a process for preparing anegatively charged microporous membrane comprising a porous substrateand a crosslinked coating having pendant anionic group. An embodiment ofthe process comprises:

[0043] (a) providing a porous substrate;

[0044] (b) contacting the substrate with a hydroxyl-rich material and apolymerized composition comprising an unsaturated monomer having ananionic group, at least one or more N-(hydroxyalkyl)- and/orN-(alkoxyalkyl)-acrylamides, a hydrophilic unsaturated monomer, andoptionally an initiator;

[0045] (c) curing the substrate obtained in (b) to obtain the negativelycharged membrane; and

[0046] (d) optionally, extracting the membrane obtained in (c) to removeextractable residue therein.

[0047] The present invention further provides a negatively chargedmembrane comprising a porous substrate and a crosslinked coating. Anembodiment of the process comprises:

[0048] (a) providing a porous substrate;

[0049] (b) contacting the substrate with a polysaccharide and apolymerized composition comprising an unsaturated monomer having ananionic group, an N-(hydroxymethyl)- and/orN-(alkoxymethyl)-acrylamides, and an initiator;

[0050] (c) curing the substrate obtained in (b) to obtain the negativelycharged membrane; and

[0051] (d) optionally, extracting the membrane obtained in (c) to removeextractable residue therein.

[0052] The present invention further provides, in an embodiment, anegatively charged microporous membrane comprising a porous support anda crosslinked coating wherein the crosslinked coating is prepared from apolymerized composition comprising an unsaturated monomer having ananionic group, an N-(hydroxymethyl)- or N-(alkoxymethyl)-acrylamide, anonionic hydrophilic acrylic monomer, and an initiator.

[0053] The present invention further provides, in another embodiment, anegatively charged microporous membrane comprising a porous substrateand a crosslinked coating prepared from a polysaccharide and apolymerized composition comprising an unsaturated monomer having ananionic group, an N-(hydroxymethyl)- or N-(alkoxymethyl)-acrylamide, andan initiator.

[0054] The present invention, in a further embodiment, provides anegatively charged microporous membrane comprising a porous substrateand a crosslinked coating prepared from a composition comprising anacrylic monomer having an anionic group, an N-(hydroxymethyl)- orN-(alkoxymethyl)-acrylamide, a nonionic Hydrophilic acrylic monomer, andan initiator.

[0055] The present invention, in another embodiment, provides anegatively charged microporous membrane comprising a porous substrateand a crosslinked coating prepared from a polysaccharide and apolymerized composition comprising an acrylic monomer having an anionicgroup, an N-(hydroxymethyl)-or N-(alkoxymethyl)-acrylamide, and aninitiator.

[0056] The present invention further provides a device e.g., a filterdevice, chromatography device, macromolecular transfer device, flowdistributor arrangement, and/or a membrane module comprising one or morenegatively charged membranes of the present invention. The device can bein any suitable form. For example, the device can include a filterelement comprising the negatively charged membrane in a substantiallyplanar or pleated form. In an embodiment, the element can have a hollowgenerally cylindrical form. If desired, the device can include thefilter element in combination with upstream and/or downstream support ordrainage layers. The device can include a plurality of membranes, e.g.,to provide a multilayered filter element, or stacked to provide amembrane module, such as a membrane module for use in membranechromatography. Filter cartridges can be constructed by including ahousing and endcaps to provide fluid seal as well as at least one inletand at least one outlet. The devices can be constructed to operate incrossflow or tangential flow mode as well as dead-end mode. Accordingly,the fluid to be treated can be passed, for example, tangentially to themembrane surface, or passed perpendicular to the membrane surface.

[0057] For embodiments of the membrane which are in the form of a tubeor fiber, or bundles of tubes or fibers, the membrane can be configuredas modules, e.g., after potting their ends with an adhesive. For adescription of illustrative chromatographic devices, porous mediummodules, and flow distributor arrangements, see U.S. Provisional PatentApplication Nos. 60/121,667 and 60/121,701, both filed on Feb. 25, 1999;U.S. Provisional Patent Application Nos. 60/168,738 and 60/168,750, bothfiled on Dec. 6, 1999 and International Applications filed on Feb. 25,2000 and entitled “Positively Charged Membrane” by Xiaosong Wu,Chung-Jen Hou, Jayesh Dharia, Peter Konstantin, and Yujing Yang;“Chromatography Devices and Flow Distributor Arrangements Used inChromatography Devices” by Mark Hurwitz, Thomas Sorensen, John Stempel,and Thomas Fendya; and “Chromatography Devices, Porous Medium ModulesUsed in Chromatography Devices and Methods for Making Porous MediumModules” by Mark Hurwitz, Thomas Fendya, and Gary Bush. See also UKPatent Application GB 2 275 626 A.

[0058] The membrane of the present invention has one or moreadvantageous properties, including high water permeability dynamicprotein binding capacity, and charge density. Thus, for example, themembrane preferably has a water flow rate above 5 mL/min/cm², andpreferably above 10 mL/min/cm², e.g., from about 20 mL/min/cm² to about160 mL/min/cm², and preferably from about 25 mL/min/cm² to about 100mL/min/cm² at 24 inch Hg. The membrane is robust and can withstand hightreatment fluid flow rates. Thus, the membrane can be subjected to flowrates up to 10 cm/min, for example, from about 1 cm/min to 10 cm/min at10 psi. The membrane has an open water bubble point of below about 70psi, e.g., from about 2.5 psi to about 70 psi, and preferably from about5 psi to about 50 psi.

[0059] The membrane of the present invention has a high charge density.The charge density of the membrane can be measured by methods known tothose of ordinary skill in the art. For example, the charge density canbe measured by the membrane's ability to bind a positively charged dye.Illustratively, the membrane has a Methylene Blue dye binding capacityof at least about 10 mL, e.g., from about 10 mL to about 1000 mL, andpreferably from about 100 mL to about 800 mL, when tested with a 10 ppmdye solution in water. Methylene Blue is a positively charged dye. Thedye binding capacity is measured by filtering under a 24 inch Hgnegative pressure, a 10 ppm by weight solution, pH 6.6, of MethyleneBlue dye in a membrane disc of 25 mm diameter, and monitoring the volumeof the filtrate until a trace of the dye begins to appear in thefiltrate.

[0060] The membrane of the present invention has a high specific proteinbinding capacity. The membrane has a lysozyme specific binding capacityof above 10 mg/mL, e.g., from about 10 mg/mL to about 130 mg/mL andpreferably from about 25 mg/mL to about 120 mg/mL. The specific bindingcapacity can be determined by the following illustrative method. A fluidcontaining a lysozyme protein in 10 mM MES buffer, pH 5.5, is filteredby passing through a membrane at 1 cm/min and the concentration of theprotein in the filtrate is measured as a function of time. Theconcentration of the protein can be determined spectrophotometrically,e.g., by measuring the absorbance of the protein at 280 nm. Abreakthrough curve such as the one shown in FIG. 1 can then beconstructed with the x-axis depicting the time of the filtrationexperiment and the y-axis depicting the protein concentration in thefiltrate. The membrane has high specific protein binding capacity andlow non-specific or hydrophobic binding. The slope of the breakthroughcurve obtained on the membrane is vertical or substantially vertical.This characteristic offers improved resolution and separation ofproteins. The membrane also has high dynamic protein binding capacity.

[0061] An advantage of the membrane of the present invention is thatproteins do not leak prior to breakthrough. Another advantage of thepresent invention is that the components of the membrane are carefullychosen so that the membrane is free or substantially free of grafts orcovalent links between the coating and the substrate. The preparation ofnegatively charged membranes of the present invention involves achemistry and procedure that is relatively simple and easy to practice.

[0062] The properties of the membranes of the present invention makethem attractive for use in the detection, separation, and/orpurification of biomolecules such as proteins, amino acids, nucleicacids, and viruses. Examples of nucleic acids include modified orunmodified, synthetic or natural RNA and DNA.

[0063] The membranes of the present invention find use in variousapplications such as filtration of fluids containing positively chargedatoms, molecules, and particulates, and macromolecular transfer fromelectrophoresis gels such as the transfer of nucleic acids and proteinsfrom electrophoresis gels to an immobilizing matrix. The membrane canfind use in the separation or purification of components present inbiological fluids. Thus, for example, the membrane can find use in thepurification of human albumins from the serum, in the therapeuticfractionation of blood, and separation of the components in geneticallyengineered cell cultures or fermentation broths. The membrane can beused in the purification of, for example, viral vaccines and genetherapy vectors such as adeno-associated viruses.

[0064] Accordingly, the present invention provides a process fortreating a fluid containing biomolecules, the process comprising placingthe fluid in contact with the negatively charged membrane. Thepositively charged materials adsorbed on the membrane can be recoveredby eluting with a suitable solvent eluant. The present invention furtherprovides a process for selectively adsorbing one or more biomoleculesfrom fluid containing a mixture of biomolecules comprising contactingthe fluid with the membrane under conditions favorable to the adsorptionof selected biomolecules. The present invention further provides aprocess for selectively releasing one or more biomolecules from amembrane of the present invention comprising contacting the membranehaving adsorbed biomolecules with an eluant under conditions favorableto the release of the selected biomolecules. The present inventionfurther provides a process for macromolecular transfer from anelectrophoresis gel comprising contacting a membrane of the presentinvention with the electrophoresis gel, and transferring themacromolecules from the gel to the membrane.

[0065] The negatively charged membrane of the present invention isparticularly suitable for treating fluids containing biomolecules thathave a positive surface charge for the given pH of the fluid. Forexample, lysozyme has an isoelectric point of 11.25, and it can bepurified by using the negatively charged membrane of the presentinvention from a low salinity, for example 10 mM MES, fluid that is pH5.5. Proteins with different surface charges may also be separated usingthe membrane of the present invention, for example separating lysozymefrom Cytochrome C.

[0066] Thus, a mixture of lysozyme and Cytochrome C can be separated asfollows. 80 μl of a fluid containing 3 mg/ml lysozyme and 1 Cytochrome Ccan be placed on a chromatographic column or stack of 5 layers of a 25mm diameter negatively charged membrane of the present invention. Thecolumn or stack can be eluted under a gradient—7 ml from 10 mM MESbuffer at a pH of 5.5 to 1M NaCl-10 mM MES buffer at a pH of 5.5. Theflow rate can be 4 ml/min. Cytochrome C elutes first, followed bylysozyme.

[0067] The following examples further illustrate the present inventionbut should not be construed in any way limiting the scope of theinvention.

EXAMPLE 1

[0068] This Example illustrates a method of preparing a polymercomposition for preparing an embodiment of the negatively chargedmembrane of the present invention.

[0069] 2-Acrylamido-2-methyl-1-propanesulfonic acid,N-(isobutoxymethyl)acrylamide, and hydroxypropyl methacrylate werecombined in a weight ratio of 8.0:2.5:1.5 in a methanol-water medium toobtain a polymerization solution having a solids content of 12% byweight. Ammonium persulfate was used as the initiator at 0.3% by weightof the solution. The polymerization was carried out for a period ofabout 10-15 hours at ambient temperature (20-25° C.). The resultingsolution had a viscosity of 166 cps.

EXAMPLE 2

[0070] This Example illustrates a method for preparing an embodiment ofthe negatively charged membrane of the present invention. This Examplefurther illustrates the properties of that embodiment.

[0071] A coating solution was prepared by mixing the polymerizationsolution described in Example 1 and a water solution of dextran,molecular weight 148 K, so that the resulting solution contains polymerand dextran in the weight ratio of 3:1.

[0072] A hydrophilic microporous polyethersulfone substrate having apore size of about 0.8 μm was coated with the above coating solution.The coated substrate was cured in an oven at 100-110° C. for 1 hour,followed by washing it in boiling DI water for 1 hour. The resultingmembrane was dried in an oven to obtain an embodiment of the presentinvention.

[0073] The membrane obtained above was tested for treatment of asolution containing lysozyme. The solution was contained 206.4 μg per mlof 10 mM MES buffer at pH 5.5. The treatment fluid flow rate was 4ml/min. Two membrane discs of 25 mm diameter were stacked together. Thebreakthrough curve obtained is set forth in FIG. 1. The membrane had alysozyme binding capacity of 97 mg/ml. The relatively flat curveobtained during the first 10 minutes of the treatment confirmed that themembrane did not leak. The nearly vertical slope indicates that themembrane was capable of providing high resolution.

EXAMPLE 3

[0074] This Example illustrates a method for preparing an embodiment ofthe negatively charged membrane of the present invention. This Examplefurther illustrates the properties of that embodiment.

[0075] A coating solution was prepared by mixing the polymerizationsolution described in Example 1 and a water solution of dextran,molecular weight 148 K, so that the resulting solution contains polymerand dextran in the weight ratio of 4:1.

[0076] A hydrophilic microporous cellulose nitrate substrate having apore size of about 0.8 μm was coated with the above coating solution.The coated substrate was cured in an oven at 100-110° C. for 1 hour,followed by washing it in boiling DI water for 1 hour. The resultingmembrane was dried in an oven to obtain an embodiment of the presentinvention.

[0077] The membrane obtained above was tested with a solution containinglysozyme. The solution was contained 201.3 μg per ml of 10 mM MES bufferat pH 5.5. The treatment fluid flow rate was 4 ml/min. Two membranediscs of 25 mm diameter were stacked together. The breakthrough curveobtained is set forth in FIG. 2. The membrane had a lysozyme bindingcapacity of 77 mg/ml. The relatively flat curve obtained during thefirst 10 minutes of the treatment confirmed that the membrane did notleak. The nearly vertical slope indicates that the membrane was capableof providing high resolution.

EXAMPLE 4

[0078] This Example illustrates a method for preparing anotherembodiment of the negatively charged membrane of the present invention.This Example further illustrates the properties of that embodiment.

[0079] 2-Acrylamidoglycolic acid, 2-carboxyethyl acrylate,N-(isobutoxymethyl)acrylamide, N-(hydroxymethyl)-acrylamide, andhydroxypropyl acrylate were combined in a weight ratio of5.0:5.0:3.0:1.5:1.5 in a methanol-water medium to obtain apolymerization solution having a solids content of 16% by weight.Ammonium persulfate was used as the initiator at 0.4% by weight of thesolution. The polymerization was carried out for a period of about 16-24hours at ambient temperature. The resulting solution had a viscosity of116 cps. A coating solution was prepared by mixing the polymerizationsolution and a water solution of dextran, molecular weight 148 K, sothat the resulting solution contained 4% polymer and 1.33% dextran byweight.

[0080] A hydrophilic microporous polyethersulfone substrate having apore size of about 0.8 μm was coated with the above coating solution.The coated substrate was cured in an oven at 100-110° C. for 1 hour,followed by washing it in boiling DI water for 1 hour. The resultingmembrane was dried in an oven to obtain another embodiment of thepresent invention.

[0081] The membrane obtained above was tested with a solution containinglysozyme. The solution was contained 213.6 μg per ml of 10 mM MES bufferat pH 5.5. The treatment fluid flow rate was 4 ml/min. Two membranediscs of 25 mm diameter were stacked together. The breakthrough curveobtained is set forth in FIG. 3. The membrane had a lysozyme bindingcapacity of 45 mg/ml. The relatively flat curve obtained during thefirst 10 minutes of the treatment confirmed that the membrane did notleak. The nearly vertical slope indicates that the membrane was capableof providing high resolution.

[0082] All references cited herein, including patents, patentapplications, and publications, are incorporated by reference in theirentireties.

[0083] While this invention has been described with an emphasis uponseveral embodiments, it will be obvious to those of ordinary skill inthe art that variations of the embodiments may be used and that it isintended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the following claims.

What is claimed is:
 1. A filter element comprising at least onenegatively charged microporous membrane comprising a porous substrateand a crosslinked coating, wherein the crosslinked coating is preparedfrom a solution comprising a polysaccharide and an anionic polymer, theanionic polymer being obtained by polymerizing a mixture comprising anunsaturated monomer having a negatively charged group, a hydrophilicnon-ionic unsaturated monomer, and at least one or moreN-(hydroxyalkyl)- or N-(alkoxyalkyl)-acrylamide monomers, wherein thecrosslinked coating comprises amide-ester and amide-amide crosslinks. 2.The filter element of claim 1, wherein the negatively chargedmicroporous membrane comprises a pleated membrane.
 3. The filter elementof claim 1, having a hollow, generally cylindrical form.
 4. The filterelement of claim 2, comprising a plurality of pleated negatively chargedmicroporous membranes.
 5. The filter element of claim 2, having ahollow, generally cylindrical form.
 6. The filter element of claim 4,having a hollow, generally cylindrical form.
 7. The filter element ofclaim 1, comprising a plurality of negatively charged microporousmembranes.
 8. The filter element of claim 7, having a hollow, generallycylindrical form.
 9. A device comprising a housing comprising at leastone inlet and at least one outlet and defining a fluid flow path betweenthe inlet and the outlet, and, interposed between the inlet and theoutlet and across the fluid flow path, at least one negatively chargedmicroporous membrane comprising a porous substrate and a crosslinkedcoating, wherein the crosslinked coating is prepared from a solutioncomprising a polysaccharide and an anionic polymer, the anionic polymerbeing obtained by polymerizing a mixture comprising an unsaturatedmonomer having a negatively charged group, a hydrophilic non-ionicunsaturated monomer, and at least one or more N-(hydroxyalkyl)- orN-(alkoxyalkyl)-acrylamide monomers, wherein the crosslinked coatingcomprises amide-ester and amide-amide crosslinks.
 10. A device fortangential flow filtration comprising a housing comprising an inlet anda first outlet and a second outlet; the device defining a first fluidflow path between the inlet and the first outlet, and defining a secondfluid flow path between the inlet and the second outlet, and, interposedbetween the inlet and the first outlet and across the first fluid flowpath; and, interposed between the inlet and the first outlet and acrossthe first fluid flow path, and interposed between the inlet and thesecond outlet and tangential to the second fluid flow path, at least onenegatively charged microporous membrane comprising a porous substrateand a crosslinked coating, wherein the crosslinked coating is preparedfrom a solution comprising a polysaccharide and an anionic polymer, theanionic polymer being obtained by polymerizing a mixture comprising anunsaturated monomer having a negatively charged group, a hydrophilicnon-ionic unsaturated monomer, and at least one or moreN-(hydroxyalkyl)- or N-(alkoxyalkyl)-acrylamide monomers, wherein thecrosslinked coating comprises amide-ester and amide-amide crosslinks.11. The device of claim 9, comprising a plurality of negatively chargedmicroporous membranes, each membrane comprising a porous substrate and acrosslinked coating, wherein the crosslinked coating is prepared from asolution comprising a polysaccharide and an anionic polymer, the anionicpolymer being obtained by polymerizing a mixture comprising anunsaturated monomer having a negatively charged group, a hydrophilicnon-ionic unsaturated monomer, and at least one or moreN-(hydroxyalkyl)- or N-(alkoxyalkyl)-acrylamide monomers, wherein thecrosslinked coating comprises amide-ester and amide-amide crosslinks,interposed between the inlet and the outlet and across the fluid flowpath.
 12. The device of claim 9, wherein the negatively chargedmicroporous membrane comprises a pleated membrane.
 13. The device ofclaim 9, including a filter element having a hollow, generallycylindrical form, wherein the filter element comprises the at least onenegatively charged microporous membrane.
 14. The device of claim 11,wherein the plurality of negatively charged microporous membranescomprise pleated membranes.
 15. The device of claim 11, including afilter element having a hollow, generally cylindrical form, wherein thefilter element comprises the plurality of negatively charged microporousmembranes.
 16. The device of claim 15, wherein the plurality ofnegatively charged microporous membranes comprise pleated membranes.